Cytidine triphosphate promotes efficient ParB-dependent DNA condensation by facilitating one-dimensional spreading fromparS

Faithful segregation of bacterial chromosomes relies on the ParABS partitioning system and the SMC complex. In this work, we used single molecule techniques to investigate the role of cytidine triphosphate (CTP) binding and hydrolysis in the critical interaction between centromere-likeparSDNA sequences and the ParB CTPase. Using a combined dual optical tweezers confocal microscope, we observe the specific interaction of ParB withparSdirectly. Binding aroundparSis enhanced 4-fold by the presence of CTP or the non-hydrolysable analogue CTPγS. However, ParB proteins are also detected at a lower density in distal non-specific regions of DNA. This requires the presence of aparSloading site and is prevented by roadblocks on DNA, consistent with one dimensional diffusion by a sliding clamp. Magnetic tweezers experiments show that the spreading activity, which has an absolute requirement for CTP binding but not hydrolysis, results in the condensation ofparS-containing DNA molecules at low nanomolar protein concentrations. We propose a model in which ParB-CTP-Mg²⁺complexes move along DNA following loading atparSsites and protein:protein interactions result in the localised condensation of DNA within ParB networks.